The polyene macrolide antibiotic natamycin (Antibiotic A-5283) is commonly used to retard the growth of surface molds on various cheese varieties. Natamycin is commonly applied to the surface of cheese by dipping or spraying, using an aqueous dispersion containing 200 to 300 ppm of the additive. The large molecular weight of natamycin, 666 g/mol, and conjugated double bond structure causes it to be extremely insoluble in water and most food grade solvents. The inability to apply natamycin in true solution creates void non-treated areas on the food surface. These non-treated areas promote the growth of fungal organisms. A water soluble N-alkyl semisynthetic derivative of natamycin was synthesized by the Michael addition reaction of the parent with a N-substituted malemide. A comparative study investigating the effectiveness of the semisynthetic derivative of natamycin and the parent antibiotic in suppressing mold growth on one month aged shredded Cheddar cheese modified atmosphere packaged (MAP) was performed. A 20 ppm natamycin treatment effectively suppressed visible mold growth (<104 CFU/g) in MAP samples for up to 30 days after opening. The 20 ppm semisynthetic derivative performed similarly to the 10 ppm natamycin treatment in retarding mold growth. Visible mold growth did not occur for these treatments in MAP samples until 20 days after opening. Analysis of storage conditions revealed that an outgrowth of mold in shredded cheese occurred in MAP packages stored longer than 15 days. This bloom in mold growth was attributed to the degradation of natamycin and the semisynthetic derivative throughout storage. The stability and degradation of natamycin and the derivative were monitored throughout the study. Antibiotic concentration on the cheese surface was quantified by molecular absorption spectrometry. Results from this study showed, heavily contaminated samples caused the rate and loss of natamycin and the derivative to increase. Antibiotic concentration decreased at a similar rate in MAP and open package conditions. Natamycin and derivative were found to have similar degradation properties. / Master of Science

Soybean is an important source of protein in animal feed, and growing demand for meat consumption worldwide has led to increased soybean production. Over 120 million metric tons of soybean were harvested in the United States in 2018, approximately one-third of the world production. In the Mid-Atlantic region, soybean is one of the most valuable field crops. Major foliar diseases that reduce soybean yield in the Mid-Atlantic region are frogeye leaf spot (FLS) and Cercospora leaf blight. In addition to crop rotation and host resistance, foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage these soybean foliar diseases. Yield benefits of foliar fungicides have been inconsistent and this may be the result of low disease pressure, unfavorable environmental conditions for disease development, or the presence of fungal pathogen populations that have developed resistance to fungicides. The objectives of this research were 1) to develop a pyrosequencing-based assay to rapidly quantify QoI resistance frequencies in Cercospora sojina, the causal agent of FLS, 2) to examine the effects of fungicide application timings, disease pressure, and environmental factors on soybean yield, and 3) to develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. using a threshold decision rule. A pyrosequencing assay targeting the G143A mutation was designed, and a Virginia survey of C. sojina populations indicated that the G143A mutation conferring QoI resistance is widespread. In small plot fungicide application timing experiments, five weekly fungicide applications starting at beginning pod (R3) resulted in the greatest yield, but for single fungicide applications, R3 or 1 week after R3 resulted in the greatest yields. There was positive relationship between the cumulative number of disease favorable days (mean daily temperature 20-30°C and ≥ 10 hours of relative humidity >90%) from planting to R3 and disease severity at the full pod stage (r = 0.97, P = <0.01). Higher disease severity was associated with greater yield loss (r2 =0.53, P = 0.10) suggesting foliar fungicide applications are more likely to have yield benefits as the number of disease favorable days prior to R3 increase. A disease favorable-days threshold (FDT) using the environmental parameters indicated above was evaluated in on-farm experiments throughout Virginia, Maryland, and Delaware. Based on decision rules, FDT = 8 three weeks prior to R3 was the best predictor of a yield benefit with an R3 fungicide application. The decision aid was also able to correctly predict when a fungicide application would not be profitable ≥90% of the time. This weather-based decision aid along with monitoring of fungicide resistance development within the region will provide soybean growers in the Mid-Atlantic U.S. with tools to maximize yields and profitability. / Doctor of Philosophy / Soybean is the third most valuable field crop in the world, ranked only behind rice and wheat in value. Over 98% of the soybean crop is used for animal feed due to its high protein content. The United States is the largest soybean producer in the world, responsible for one-third of global production. Soybean is the top cash crop in the Mid-Atlantic region. Foliar fungal diseases can reduce the soybean yield by causing lesions on the leaves that reduce photosynthesis and cause premature defoliation. Frogeye leaf spot (FLS) caused by Cercospora sojina is a major yield reducing soybean foliar diseases in the Mid-Atlantic region. Foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage the disease. However, fungicide efficacy has been inconsistent. Inconsistencies may be due to low disease pressure, improper application timing, or fungicide resistance. The purpose of this research was to investigate the fungicide efficacy inconsistencies and to develop management tools to improve yield and maximize profitability. Our objectives were to 1) develop a molecular assay to quantify frequencies of the mutation conferring fungicide resistance in Virginia populations of C. sojina, 2) examine the effects of fungicide application timings, disease severity, and weather on soybean yield, and 3) develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. The C. sojina fungicide resistance mutation was widespread in Virginia, but overall frequencies were relatively low compared to findings from Midwest and Southern states. In fungicide timing experiments, beginning pod (R3) applications resulted in the most consistent yield benefits, and disease severity and yield loss increased as the number of weather-based disease favorable days prior to R3 increased. We used data from on-farm experiments in Virginia, Maryland, and Delaware to develop a weather-based disease favorable-days threshold that increased the probability that a fungicide application at R3 would have a yield benefit in soybean. The results of our research have led improved fungal disease management recommendations for soybean in the Mid-Atlantic that will maximize yields and profitability.

Colletotrichum acutatum and C. gloeosporioides are fungal plant pathogens that have a global distribution, extensive host range, and convoluted taxonomy. Both species can cause grape ripe rot and are considered endemic to Virginia US. In 2012, C. acutatum and C. gloeosporioides were reclassified into species complexes that consist of 31 and 22 accepted species, respectively. The objectives of this study were to: 1) survey Virginia vineyards for grape ripe rot, and morphologically and phylogenetically identify isolates to the species within the complexes, 2) conduct an in vitro fungicide assay to screen fifteen commercial fungicides and combinations of two fungicides for efficacy to control isolates from seven Colletotrichum species from Virginia vineyards, 3) sequence gene fragments from three subunits of the SDH enzyme in the fungicide-screened isolates to observe potential resistance mutations, 4) investigate the susceptibility of three grapevine tissues to Colletotrichum species, 5) observe potential infection structures before and after the application of fungicides, 6) evaluate the efficacy of commercial fungicide controls of grape ripe rot in the field, and determine the most advantageous timing of applications. In my studies, I identified six Colletotrichum species: C. aenigma, C. conoides, C. fioriniae, C. gloeosporioides, C. kahawae, and C. nymphaeae. I also found two additional groups; an isolate similar to C. limetticola and C. melonis and a group of isolates that are similar to C. alienum, C. fructicola, and C. nupharicola. I also identified captan, and mancozeb as two potential active ingredients for control of grape ripe rot isolates from Virginia via the in vitro fungicide assay. Additionally, I found that combinations of two active ingredients could increase the efficacy of benzovindiflupyr, copper, and polyoxin-D. C. fioriniae germination and production of melanized appressoria was documented on leaves. I observed appressorium formation with isolates of two C. fructicola-like genotypes and C. nymphaeae, as well as secondary conidiation with isolates of C. aenigma, C. fructicola-like genotype 3, and C. nymphaeae on blooms. And finally, benzovindiflupyr, cyprodinil + fludioxonil pre-mix, and potassium phosphite + tebuconazole were identified as candidates for chemical control for grape ripe rot in the field. / PHD / Colletotrichum acutatum and C. gloeosporioides are two fungal plant pathogens that are found on a wide range of crops around the globe. Both fungal species cause the disease grape ripe rot and have been found in Virginia (VA) USA since the late 1800s. Originally, grape ripe rot was considered a minor disease in VA; however, based on communications with local VA vineyard managers, grape ripe rot was found to cause up to 30% direct crop loss. Further indirect economic loss occurs during wine production due to the production of unpalatable, tobacco-like, off flavors from the infected grapes. Sensory studies found this wine flavor change occurred with as little as 3% of the total crushed grapes being infected. Grape ripe rot appears as a sunburn-like, tan injury on the surface of white-fruited grape berries. As the disease progresses, the dark injury expands across the surface of the berry and rings of salmon-colored spore masses form. On red-fruited grapes, the formation of spore masses is usually the first observable sign. Over time, the infected berries will shrivel down to a soft, pustule-covered raisin. Both C. acutatum and C. gloeosporioides cause the same grape ripe rot symptoms on fruit and overlap in fungal appearance. In addition, investigations of these pathogens using molecular techniques have revealed that each consists of a number of genetically distinct groups that are difficult to distinguish by appearance. Therefore, in 2012, C. acutatum and C. gloeosporioides were reclassified into 31 and 22 newly accepted species, respectively, using molecular techniques. The objectives of this study were to: 1) survey VA vineyards for grape ripe rot, and visually and molecularly identify isolates to the species within the new complexes, 2) conduct a laboratory fungicide assay to screen fifteen commercial fungicides and combinations of two fungicides for control of isolates from VA vineyards, 3) sequence gene fragments from three subunits of the SDH enzyme in the fungicide-screened isolates to observe potential resistance mutations, 4) investigate the susceptibility of three grapevine tissues to Colletotrichum species, 5) observe potential infection structures before and after the application of fungicides, 6) evaluate the efficacy of commercial fungicide controls of grape ripe rot in the field, and determine the most advantageous timing of applications. In my studies, I identified six Colletotrichum species: C. aenigma, C. conoides, C. fioriniae, C. gloeosporioides¸ C. kahawae, and C. nymphaeae. I also found two additional groups; an isolate similar to C. limetticola and C. melonis and a group of isolates that are similar to C. alienum, C. fructicola, and C. nupharicola. Our lab also identified four active ingredients as potential controls of grape ripe rot in the laboratory fungicide assay; captan, mancozeb, tetraconazole and thiophanate-methyl. Additionally, combinations of two compounds can increase the effectiveness of benzovindiflupyr, copper, and polyoxin-D. of C. fructicola-like isolates, and C. nymphaeae formed infection structures on blooms. of C. aenigma, C. fructicola-like genotype 3, and C. nymphaeae formed spores on blooms without producing symptoms. C. fioriniae spores germinated and produced infection structures on leaves without producing symptoms.

<p dir="ltr">In response to fluctuating corn (<i>Zea mays</i> L.) prices, climatic variability, and emerging diseases, farmers are increasingly adopting diverse and intensive management practices to enhance yield and profitability. This research investigates the performance of various inputs and management practices on corn production across multiple site-years, with a focus on yield components, grain fill duration, kernel development, disease severity, and economic outcomes. A multi-state research trial was established to evaluate the impact of seven inputs and management practices across multiple locations and environments in Indiana, Kentucky, and Michigan in 2022 and 2023. Each location included eight treatments: 1) control treatment (C) based on Purdue University seed rate and nitrogen (N) fertilizer recommendations (Camberato et al., 2022; Nielsen et al., 2022): 30K seeds per acre and N fertilizer application as starter (2x2) and V5 growth stage sidedress. Total N rates ranged between 180 and 200 lbs N per acre and agronomic optimum nitrogen rates (AONR) were used, 2) C + banded (2x2) fungicide, 3) C + 20% increase in corn seeding rate, 4) C + sulfur (S) fertilizer, 5) C + foliar micronutrients, 6) C + late-season N fertilizer application (V10-12 growth stage), 7) C + R1 foliar fungicide, and 8) intensive treatment (all additional inputs/management practices applied). The intensive treatment significantly increased yield by 16.4 and 18.4 bu ac^-1 in 2022 and 2023, respectively when compared to the control across locations, but did not enhance net profit across multiple corn price scenarios due to high application costs. Conversely, R1 fungicide applications increased yield by 16.2 and 16.7 bu ac^-1 in 2022 and 2023, respectively, and S applications increased yield by 12.9 bu ac^-1 in 2023, when compared to the control, with both treatments improving net profit under multiple corn price scenarios. In addition, kernel development studies in West Lafayette, IN, during 2022 and 2023 revealed that banded fungicide applications at planting and foliar fungicide applications at the R1 growth stage can reduce leaf disease severity by 3.2% to 6.6%, extend grain fill duration by 3.5 to 4.5 days, and increase maximum dry kernel weight at plant maturity by 5.7 to 9.4%, respectively, leading to further insights into the yield response mechanisms. Furthermore, a meta-analysis of 24 at-plant flutriafol fungicide placement trials across Indiana (2020 – 2023) highlighted the effectiveness of at-plant fungicides, with banded (2x2 or 2x0) applications leading to the highest yield increase of 7.8 bu ac^-1 and both banded and in-furrow applications reducing disease severity on corn ear leaves at the R5 growth stage by 2.1 - 2.3% when compared to the control. These findings suggest both at-plant banded and R1 foliar fungicide applications have the potential to reduce disease severity, extend corn grain fill duration, and improve yield when conditions are conducive for a response (e.g., foliar disease presence). Overall, this research highlights the ability of targeted input applications for improving both corn yield and profitability when examined across diverse environments and locations, rather than prophylactic applications of multiple inputs and increased management intensities.</p>

Thesis (MScAgric)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry. / AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.

Master of Science / Department of Plant Pathology / Megan Kennelly / Dollar spot disease of turfgrass, caused by the fungus Sclerotinia homoeocarpa, is the most economically important disease of intensively managed turfgrass such as creeping bentgrass (Agrostis stolonifera) in golf course fairways and putting greens. While several cultural management practices can lessen the severity of the disease, fungicide applications are necessary to manage the disease to acceptable levels. Host resistance is another avenue of improving the quality of turfgrass in response to dollar spot disease, but more information on cultivar susceptibility levels is necessary. Many fungicides are available to control dollar spot, but S. homoeocarpa has shown resistance to both demethylation inhibitor (DMI) and benzimidazole class fungicides. The objectives of this thesis were: (1) to evaluate 15 cultivars of creeping bentgrass under putting green and fairway management for both overall quality and disease severity of dollar spot in the presence and absence of fungicide applications; (2) to determine the sensitivity of Kansas S. homoeocarpa isolates to the DMI fungicides propiconazole, metconazole, tebuconazole, and triticonazole using in vitro mycelia growth assays; (3 to determine a best discriminatory dose concentration for each DMI fungicide that will provide for rapid testing of relative sensitivity, and (4) to sequence the entire β-tubulin gene of several resistant and sensitive isolates to determine mutations associated with resistance. There were differences in turfgrass quality among the 15 cultivars at putting green height but not fairway height. There were no significant differences in disease. In the fungicide resistance assays, the mean EC50 values were 0.0163, 0.038, 0.0612, and 0.0994 µg/ml for metconazole, propiconazole, tebuconazole, and triticonazole, respectively. Correlations were significant and positive for all pairwise comparisons of log10EC50 values. Regressions using discriminatory concentrations tested were significant. The most predictive concentrations were 0.01, 0.05, 0.05, and 0.10 µg/ml a.i, for metconazole, propiconazole, tebuconazole, and triticonazole, respectively. The entire β-tubulin gene was sequenced for four resistant and four sensitive isolates. The resistant isolates all harbored a substitution of alanine for glutamic acid at codon 198 (E198A).

Phoma stem canker is a disease of oilseed rape (Brassica napus) caused by closely related plant pathogens Leptosphaeria maculans and L. biglobosa. It is an economically important disease, causing annual yield losses of approximately £770 million worldwide. When colonising oilseed rape, L. maculans and L. biglobosa exist in close proximity on the leaf, competing for resources as they move through the main leaf vein and into the stem. Fungicides are commonly used to decrease severity of phoma stem canker on oilseed rape. However, the efficacy and longevity of active chemicals is under threat from evolution of resistance in pathogen populations and government legalisation. Moreover, it has been suggested that both L. maculans and L. biglobosa differ in their sensitivity to azoles, and important class of fungicides that are used to control the disease through the inhibition of lanosterol 14-α demethylase (erg11, CYP51). This project aims to further understand the role that fungicides have in controlling phoma stem canker by investigating their efficacy against L. maculans and L. biglobosa in crops, in vitro and in planta. In field experiments, established in Cambridgeshire across four cropping seasons, the fungicide mixture penthiopyrad (SDHI) plus picoxystrobin (QoI) was as effective at controlling phoma leaf spotting and phoma stem canker in winter oilseed rape as prothioconazole (DMI), suggesting that both fungicides could be used to reduce phoma stem canker symptoms. The two pathogens differed in their growth rates in vitro, with L. biglobosa growing faster than L. maculans when untreated or treated with lower fungicide concentrations. Fungicide sensitivity assays suggest that L. maculans and L. biglobosa are both sensitive to DMI, SDHI and QoI fungicides and that differences between the species are minor. Prothioconazole and penthiopyrad + picoxystrobin had a similar efficacy on oilseed rape cotyledons colonised with either L. maculans or L. biglobosa. There was no difference between species on prothioconazole treated plants, although there was a difference between L. maculans and L. biglobosa when treated with 20 μg/ml penthiopyrad + picoxystrobin. Heterologous yeast expression of LmCYP51B and LbCYP51B with fungicide sensitivity testing of the yeast transformants suggests that LmCYP51B and LbCYP51B are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. These findings are supported by homology protein modelling, which predicts that LmCYP51B and LbCYP51B are structurally very similar, specifically at the azole-binding site. In conclusion, fungicides are still an effective control method for reducing phoma stem canker symptoms caused by Leptosphaeria species in the UK, and a useful tool to in the sustainable production of oilseed rape.

Orientador: Antonio de Goes / Resumo: A ferrugem alaranjada da cana-de-açúcar (FA), causada por Puccinia kuehnii, é responsável por superior a 40% na produtividade de genótipos de cana-de-açúcar suscetíveis e intermediários. Nesse estudo foram realizados três experimentos. No experimento E1 foi avaliado o efeito do número de aplicações de fungicida piraclostrobina + epoxiconazole (PE) no município de Olímpia-SP, em E2 foi avaliado o efeito do número de aplicações de fungicida piraclostrobina + epoxiconazole (PE) no município de Catigua-SP, ambos em épocas distintas, e em E3 foi avaliado o efeito do volume de calda de PE no controle da doença. A partir dos dados obtidos nos experimentos E1 e E2, foi também avaliada a viabilidade econômica do controle da FA da cana-de-açúcar. Os experimentos foram realizados em dois locais, Olímpia e Catiguá, Estado de São Paulo, sendo empregada a cultivar SP81-3250, em quarto ciclo, após o terceiro corte, em estádio de desenvolvimento (Elongação do colmo; Crescimento intenso; Início do acúmulo de sacarose) da cultura. Em E1 e E2 usou-se o delineamento experimental em blocos ao acaso (DBC), com quatro tratamentos, sendo 0 (testemunha), 1, 2 e 3 aplicações. Cada parcela foi constituída por quatro linhas de 8 metros, espaçadas de 1,5 metros. Foram realizadas sete avaliações, com as quais determinou-se a severidade, a partir das quais foi obtida a área abaixo da curva de progresso da doença (AACPD). Também foi estimado o rendimento de colmos, expresso em tonelada de colmos por hectare... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The orange rust of sugarcane (FA), caused by Puccinia kuehnii, is responsible for over 40% in the yield of susceptible and intermediate sugarcane genotypes. Three experiments were performed in this study. In the E1 experiment, the effect of the number of applications of fungicide pyraclostrobin + epoxiconazole (PE) in the municipality of Olímpia-SP, in E2, was evaluated the effect of the number of applications of fungicide pyraclostrobin + epoxiconazole (PE) in the municipality of Catigua-SP, both at different times, and in E3 the effect of the volume of PE syrup on disease control was evaluated. From the data obtained in experiments E1 and E2, the economic viability of sugarcane FA control was also evaluated. The experiments were carried out at two locations, Olímpia and Catiguá, State of São Paulo, and the cultivar SP81-3250 was used, in the fourth cycle, after the third cut, at the development stage (Elongation of the stem, intense growth; sucrose) of the culture. In E1 and E2, the experimental design in randomized blocks (DBC) was used, with four treatments, being 0 (control), 1, 2 and 3 applications. Each plot was constituted by four lines of 8 meters, spaced of 1.5 meters. Seven evaluations were performed, with which the severity was determined, from which the area under the disease progress curve (AACPD) was obtained. The yield of stalks, expressed in ton of stems per hectare (TCH), was also estimated. In E3 a similar procedure was adopted, whose treatments were repres... (Complete abstract click electronic access below) / Mestre

Ambient ionization mass spectrometry is a technique used for the analysis of samples under ordinary ambient conditions. It has many advantages, such as fast sample exchange, low operation time and few sample preparation which comparing with thus ion source operating in vacuum. Therefore, several different ambient ionization sources have been developed in the past decade. Direct Electrospray Probe ionization mass spectrometry (DEP-MS) was developed by Shiea, J in 1999, which providing an effective means of analyzing not only large biomolecules, but also small organic and inorganic compounds. It can rapidly generate electrospray from a droplet which was deposited on a probe. The DEP technique provides a number of unique analytical features containing: (a) low sample consumption and sample switching is immediate; (b) the capillary and pump are unnecessary; and (c) the probe is low-cost and easy to construct and clean, and is free to maintenance. The first part of experiment in this research is based on the ideal of DEP. The leaf tissues were cut into a triangular shape, 15 mm long and 8 mm wide at the base, is held by a crocodile clip with the apex facing the inlet of the mass spectrometer and at a distance of 2 cm or more from it. Electrospray solutions are added onto the tissue to mix with the leaf sap, and a high voltage (4.5-5.5 kV) is applied to the leaf tissue through the crocodile clip, and generate ions for MS analysis. This method has been demonstrated to be applicable for the analysis of a wide variety of plant sample, such as the leaves tissue in different color, different growth, diseased and pesticide residue. The second part of experiment is improved from the first research. Electrospray was produced via a metal probe which binding two fine stainless steel needle (0.27 mm diameter) together. This probe plays the role of sampling and ionization. An amount of liquid sample was loaded onto the probe when the tip of probe touched the plant tissue, then electropray solution are introduced by syringe pump into the probe and applied high voltage on the probe to produce electrospray ionization. Therefore, direct rapid analysis was achieved using this probe interfaced and this method can direct profile of phytochemicals in a section of plant tissue in different regions, including root, stem, leaf, outer and inner part of fruit. In addition, using this probe, solid samples are directed sampling equally. The solid materials can be observed on the tip of probe by scanning electron microscope (SEM). It can obtain reasonable strong ion signals rapidly for agrochemicals deposited on the surface of vegetables or fruits peel.

Fusarium head blight (FHB) caused by Fusarium graminearum and Septoria tritici blotch (STB) caused by Septoria tritici are economically important wheat diseases in Ontario. Both reduce yield, FHB is associated with mycotoxin accumulation including deoxynivalenol (DON). Different F. graminearum chemotypes produce either DON/15-acetyldeoxynvialenol (ADON) or DON/3-ADON. The majority (97.5%) of F. graminearum isolates collected from commercial fields across Ontario were 15-ADON chemotype, the remaining were 3-ADON. In inoculated field experiments 3-ADON chemotypes were more aggressive and toxic compared with 15-ADON chemotypes as measured by FHB symptoms and DON content. In inoculated field experiments with a population derived from ‘RCATL33’ and ‘RC Strategy’ soft red winter wheat parents, genetic resistance was more effective than fungicide application at controlling FHB. Field trials with the hard red winter wheat population derived from ‘Maxine’ and ‘FTHP Redeemer’ parents revealed that STB and FHB phenotypic resistance had no negative impact on grain yield in the absence of disease.